This proposal focuses on the study of the transcription of selected Drosophila heat shock genes that are coordinately activated by heat shock yet differentially activated by normal developmental signals. We plan to further define cis-acting DNA sequence elements responsible for the heat shock and developmental activation of these genes by assaying the activity of in vitro-altered genes in germline transformants. This includes investigation of the spatial relationships and interdependence of the individual components of the heat shock and developmental regulatory responses. Such information will guide the formulation of testable mechanisms of transcriptional regulation. These studies of cis-acting regulatory elements of heat shock genes will be correlated with studies of the chromosomal configuration and chromatin structure of heat shock loci. We propose to determine the extent of the chromosomal region or domain that participates in gene activation by studies at the cytological level using a high resolution method of in situ hybridization to polytene chromosomes, and at the chromatin level by defining the region of enhanced nuclease sensitivity. At still higher resolution, we will use our newly-developed protein-DNA crosslinking method to examine in vivo interactions of specific proteins and DNA sequences and the changes in these protein-DNA associations that accompany gene activation by heat shock.